PROJECT ABSTRACT A critical gap remains in our understanding of oxygen metabolism, delivery, and reserve, both at rest and during metabolic activation states in healthy and diseased brain. Available imaging techniques lack spatial and temporal resolution to assess distribution of cerebral tissue oxygenation (PO2) and O2 consumption (CMRO2) with adequate level of detail, and to unravel their dynamic changes within microvascular domains. We propose to develop and validate a new microscopic imaging method of resting state CMRO2 in small animal models, and advance it to enable rapid high spatial resolution imaging of CMRO2 and PO2. The new technology will enable regional estimation of CMRO2 at a rate of ~1 Hz together with acquisition of detailed tissue PO2 maps, which is essential for advancing our understanding of O2 delivery and consumption in the brain. In Aim1 we will develop and validate high-resolution resting state CMRO2 imaging method in small rodents based on two- photon PO2 microscopy. This new method will measure resting state regional CMRO2 based on tissue PO2 profiles around cortical penetrating arterioles. In Aim 2 we will advance new CMRO2 imaging method by improving a multifocal, frequency modulated, two-photon phosphorescence lifetime imaging to allow practical rapid PO2 imaging in optically scattering brain tissue, and to improve the speed of CMRO2 measurements up to 100-fold (approaching 1 Hz). In Aim 3 we will quantify regional CMRO2 during physiological and pathological perturbations. We will test the hypothesis that transient physiological or pathological neuronal activation in a brain affected by ischemia leads to a mismatch between O2 metabolism and supply and hypoxia within microvascular domains - a mechanism that may underlie lesion growth in stroke and other brain injury states as well as progressive neurodegeneration observed in microvasculopathies. This technology will have broad utility in quantifying metabolism and oxygenation in cerebral microvascular domains in animal models of brain disorders that will dramatically advance our understanding of pathophysiology and lead to novel treatment strategies in important clinical problems such as chronic cerebral hypoperfusion, stroke, small vessel disease, and AD dementia.